RESUMO
Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.
Assuntos
Endossomos/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Linhagem Celular , Endossomos/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Melanócitos/citologia , Melanossomas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Qa-SNARE/genéticaRESUMO
Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.
Assuntos
Movimento Celular/fisiologia , Myxococcus xanthus/fisiologia , Esporos Bacterianos/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Citoesqueleto/fisiologiaRESUMO
ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core-ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core-ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Endocitose , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Sinais Direcionadores de Proteínas , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dimerização , Células HeLa , Humanos , Hidrólise , Células K562 , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Corpos Multivesiculares/ultraestrutura , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.
Assuntos
Diferenciação Celular , Complexo de Golgi , Proteínas da Matriz do Complexo de Golgi , Queratinócitos , Lisossomos , Lisossomos/metabolismo , Queratinócitos/metabolismo , Queratinócitos/citologia , Complexo de Golgi/metabolismo , Humanos , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas de Membrana/metabolismoRESUMO
AsiDNA is a DNA repair inhibitor mimicking DNA double-strand breaks (DSB) that was designed to disorganize DSB repair pathways to sensitize tumors to DNA damaging therapies such as radiotherapy and chemotherapy. We used the property of AsiDNA of triggering artificial DNA damage signaling to examine the activation of DSB repair pathways and to study the main steps of inhibition of DNA repair foci after irradiation. We show that, upon AsiDNA cellular uptake, cytoplasmic ATM and PARP are rapidly activated (within one hour) even in the absence of irradiation. ATM activation by AsiDNA leads to its transient autophosphorylation and sequestration in the cytoplasm, preventing the formation of ATM nuclear foci on irradiation-induced damage. In contrast, the activation of PARP did not seem to alter its ability to form DNA repair foci, but prevented 53BP1 and XRCC4 recruitment at the damage sites. In the nucleus, AsiDNA is essentially associated with DNA-PK, which triggers its activation leading to phosphorylation of H2AX all over chromatin. This pan-nuclear phosphorylation of H2AX correlates with the massive inhibition, at damage sites induced by irradiation, of the recruitment of repair enzymes involved in DSB repair by homologous recombination and nonhomologous end joining. These results highlight the interest in a new generation of DNA repair inhibitors targeting DNA damage signaling.
Assuntos
Quebras de DNA de Cadeia Dupla , Inibidores de Poli(ADP-Ribose) Polimerases , DNA , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
Methods that provide controlled influx of molecules into cells are of critical importance for uncovering cellular mechanisms, drug development and synthetic biology. However, reliable intracellular delivery without adversely affecting the cells is a major challenge. We developed a platform for on-demand intracellular delivery applications, with which cell membrane penetration is achieved by inductive heating of micro needles. The micro needles of around 1 µm in diameter and 5 µm in length are made of gold using a silicon-based micro fabrication process that provides flexibility with respect to the needles' dimensions, pitch, shell thickness and the covered area. Experiments with HCT 116 colon cancer cells showed a high biocompatibility of the gold needle platform. Transmission electron microscopy of the cell-needle interface revealed folding of the cell membrane around the needle without penetration, preventing any delivery, which was confirmed using the EthD-1 fluorescent dye. The application of an alternating magnetic field, however, resulted in the delivery of EthD-1 by localized heating of the micro needles. Fluorescence quantification showed that intracellular delivery, with as high as 75% efficiency, is achieved for specific treatment times between 1 and 5 minutes. Overexposure of the cells to the heated micro needles, i.e. longer magnetic field application, leads to an increase in cell death, which can be exploited for cleaning the platform. This method allows to perform intracellular deliver by remotely activating the micro needles via a magnetic field, and it is controlled by the application time, making it a versatile and easy to use method. The wireless actuation could also be an attractive feature for in-vivo delivery and implantable devices.
Assuntos
Citoplasma , Agulhas , Células HCT116 , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Amiloide/química , Amiloide/metabolismo , Melanossomas/metabolismo , Antígeno gp100 de Melanoma/química , Antígeno gp100 de Melanoma/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Imunofluorescência , Humanos , Imuno-Histoquímica , Melanócitos/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Interferência de RNA , RNA Interferente PequenoRESUMO
Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.
Assuntos
Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Organelas/ultraestrutura , Animais , HumanosRESUMO
CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.
RESUMO
Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.
Assuntos
Amiloide/genética , Apolipoproteínas E/genética , Melanócitos/metabolismo , Amiloide/metabolismo , Amiloide/ultraestrutura , Animais , Apolipoproteínas E/deficiência , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Endossomos/ultraestrutura , Exossomos/metabolismo , Exossomos/ultraestrutura , Regulação da Expressão Gênica , Células HeLa , Humanos , Melanócitos/ultraestrutura , Melanossomas/metabolismo , Melanossomas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
In bacteria, multicellular behaviors are regulated by cell-cell signaling through the exchange of both diffusible and contact-dependent signals. In a multicellular context, Myxococcus cells can share outer membrane (OM) materials by an unknown mechanism involving the traAB genes and gliding motility. Using live imaging, we show for the first time that transient contacts between two cells are sufficient to transfer OM materials, proteins and lipids, at high efficiency. Transfer was associated with the formation of dynamic OM tubes, strongly suggesting that transfer results from the local fusion of the OMs of two transferring cells. Last, large amounts of OM materials were released in slime trails deposited by gliding cells. Since cells tend to follow trails laid by other cells, slime-driven OM material exchange may be an important stigmergic regulation of Myxococcus social behaviors. DOI:http://dx.doi.org/10.7554/eLife.00868.001.